Morphology, Taxonomy, Anatomy, and Palynology of the Opium Poppy (Papaver somniferum L.) Cultivation in Northern Thailand

In this paper, we present the morphology, taxonomy, anatomy, and palynology of Papaver somniferum. A detailed morphological description and illustrations of the species are provided, along with information about the identification, distribution, cultivation areas, habitats, pollinators, specimens examined, growing periods, phenology, etymology, vernacular name, and uses. The species can be characterized as a glabrous and glaucous herb with unlobed or pinnately lobed leaves, and an amplexicaul base; variations in color and morphological characteristics of petals; and white filaments, occasionally purple with a white basal part, broadened at the apical part. Two rings of discontinuous and widely spaced collateral vascular bundles are recognized in the transverse section of the stems. The shape of epidermal cells on the adaxial surface is polygonal, while that on the abaxial surface is polygonal or irregular. The anticlinal cell walls of the epidermal cells on the adaxial surface are straight or slightly curved, while those on the abaxial surface are straight, slightly curved, sinuate, or strongly sinuate. The stomata are anomocytic and are confined to the lower epidermis. The stomatal density is 54–199/mm2 (with a mean of 89.29 ± 24.97). The mesophyll is not distinctly differentiated into palisade and spongy regions. Laticifers are found in the phloem areas of the stems and leaves. The pollen grains can be spheroidal or prolate spheroidal in shape, sometimes oblate spheroidal [polar axis (P) diameter/equatorial axis (E) diameter ratio = 0.99–1.12 (with a mean of 1.03 ± 0.03)]. The pollen aperture is tricolpate and the exine sculpturing is microechinate.


Introduction
Papaver L. belongs to a group of annual and perennial herbs in the subfamily Papaveroideae Eaton under the family Papaveraceae Juss., and the order Ranunculales Juss. ex Bercht. & J. Presl [1,2]. The genus consists of 149 accepted species and is distributed in the temperate and subtropical regions of the Northern Hemisphere and up to South Africa [3]. The genus is characterized as a group of herbaceous annual and perennial plants producing latex; they usually have sessile leaves, which can sometimes have an amplexicaul base; drooping flower buds; caducous sepals and petals; and petals in two whorls. The outer whorl is larger. It usually has numerous stamens; a one-locule ovary, with numerous ovules; and radiate and sessile, actinomorphic stigmas, united into a compressed, Therefore, in this paper, we provide knowledge relevant to opium poppy grown in Thailand, which was obtained from the research project entitled "Morphology, Taxonomy, Anatomy, and Palynology of the Opium Poppy (Papaver somniferum L.), Papaveraceae, Cultivation in Northern Thailand". We have obtained permission to study and support from the Narcotics Crop Survey and Control Institute, ONCB, Ministry of Justice.
Etymology: Papaver somniferum was named by Carl Linnaeus (1707-1778), a Swedish botanist, physician, and zoologist [33,34]. The generic name "Papaver" comes from a Latin name for poppies, including the opium poppy [35,36]. The specific epithet "somniferum" is a Latin word meaning sleep-inducing [36], referring to the latex exuded from the fruit, which has a sedative property of some opiate drugs.
Vernacular Name: Fin (ฝิ ่ น) (General); Breadseed poppy and Opium poppy (English). Uses: Opiate drugs derived from the milky latex found in unripe fruits (capsules) of opium poppy, which include morphine, heroin, codeine, and other alkaloids, are used in making analgesics [2,8]. Poppy seeds (mostly Papaver somniferum and P. rhoeas) are commonly used as a condiment and are a traditional ingredient in breads, bagels, and other baked goods. A paste of ground poppy seeds and sugar is used in making central and eastern European patisserie [2]. The seeds are used in food, oils, and pharmaceuticals to treat inflammation, and are cardiotonic, mildly astringent, analgesic, and sedatives [25].
Opium alkaloids (morphine, codeine, thebaine, noscapine, and papaverine) have been detected in poppy seeds and are widely used by the food industry for decoration and flavoring, but can introduce opium alkaloids into the food chain. Of the opium alkaloids found in poppy seeds, morphine and codeine are the most pharmacologically active compounds. The European Food Safety Authority has set an acute reference dose of 10 µg/kg of body weight as a safe level for morphine consumed through food products [26].

Stem Anatomy
Transverse sections of the stem of Papaver somniferum are circular outlines and consist of the epidermis, cortex, vascular bundles, and pith. The epidermal cells are covered by the cuticle. Three types of distinct tissues are found in the cortex: parenchyma, sclerenchyma, and collenchyma. Several collenchyma layers are found below the epidermis, and several sclerenchyma layers are found below the collenchyma layers. The central pith is made up of parenchyma cells. Two rings of discontinuous and widely spaced, numerous collateral vascular bundles are recognized. Laticifers are associated with the phloem tissues of the vascular bundles ( Figure 11).

Leaf Lamina Anatomy
Leaf lamina epidermis: The leaf lamina epidermis of Papaver somniferum is usually covered with wax. The epidermal cells on the adaxial surface are polygonal in shape and have straight or slightly curved anticlinal cell walls, while those on the abaxial surface are polygonal or irregular shapes and have straight, slightly curved, sinuate, or strongly sinuate anticlinal cell walls. The stomata are confined to the lower epidermis (hypostomatic leaves) and are anomocytic-type without subsidiary cells, surrounded by epidermal cells. The size of the stomata are 22.29-52.77 (with a mean of 37.50 ± 6.75) µm length, 8.11-28.43 (with a mean of 15.92 ± 3.69) µm width, and the stomatal density is 54-199/mm 2 (with a mean of 89.29 ± 24.97) (Figure 12).
Transverse section of leaf lamina: The mesophyll is dorsiventral. It is composed of 1-3 layers of palisade cells and several layers of spongy cells, but not distinctly differentiated into palisade and spongy regions in agreement with Metcalfe and Chalk [37]. The vascular bundle is solitary and surrounded by parenchyma cells. The midrib can be distinguished by a larger vascular bundle than the other veins. Laticifers are found in the phloem areas ( Figure 12).

Leaf Lamina Anatomy
Leaf lamina epidermis: The leaf lamina epidermis of Papaver somniferum is us ered with wax. The epidermal cells on the adaxial surface are polygonal in shape straight or slightly curved anticlinal cell walls, while those on the abaxial surfa lygonal or irregular shapes and have straight, slightly curved, sinuate, or strong anticlinal cell walls. The stomata are confined to the lower epidermis (hyp leaves) and are anomocytic-type without subsidiary cells, surrounded by epide The size of the stomata are 22.29-52.77 (with a mean of 37.50 ± 6.75) µm length, (with a mean of 15.92 ± 3.69) µm width, and the stomatal density is 54-199/mm mean of 89.29 ± 24.97) (Figure 12).
Transverse section of leaf lamina: The mesophyll is dorsiventral. It is compo layers of palisade cells and several layers of spongy cells, but not distinctly diff into palisade and spongy regions in agreement with Metcalfe and Chalk [37]. Th bundle is solitary and surrounded by parenchyma cells. The midrib can be dist
According to previous studies, the latex exuded from the fruit of Papaver is usually white (milky), sometimes watery, yellow, orange, or red [4,[6][7][8][9][10][11], but from our observations, we found the latex can be smelly, milky, turning pale orange or pale orange-pink, brown, and turning dark brown or black when dry.
Previously, the anomocytic (irregular-celled) type was termed ranunculaceous, which was taken from the family name Ranunculaceae, in which it was first observed [37].
From our observations, the shape and anticlinal walls of the epidermal cells on both leaf surfaces of Papaver somniferum are similar to those of P. bracteatum Lindl. and P. orientale L. from the result of Tavakkoli and Assadi [38].
According to Metcalfe and Chalke [37], the stems in the transverse section of Papaver (e.g., P. orientale) sometimes have several rings of collateral vascular bundles; furthermore, we found two rings of discontinuous and widely spaced collateral vascular bundles.
According to Bird et al. [27], the benzylisoquinoline alkaloids of opium poppy accumulate in the cytoplasm, or latex, of specialized laticifers that accompany vascular tissues throughout the plant; however, from our study, we observed them in stems and leaves.
According to Özkök and Sorkun [28], the pollen grains of Papaver somniferum are: white and purple opium poppy flowers that are similar in morphology; tricolpate in aperture, oblate spheroidal in shape (P/E ratio = 0.95), and microechinate in sculpturing. Furthermore, from our observations, we found that the shape of pollens are spheroidal or prolate spheroidal (P/E ratio = 0.99-1.12).

Materials and Methods
Plant specimens of Papaver somniferum were observed and collected in the northern region of Thailand (Mae Hong Son and Chiang Mai provinces) ( Figure A1). Herbarium specimens deposited in BK, BKF, QBG, and those included in the digital herbarium databases of BM, E, K, K-W, L, and P were examined by consulting the taxonomic literature [4][5][6][7][8][9][10][11]22,33,39] (acronyms follow those in the study by Thiers [40]). The herbarium accession number can be seen on the specimens examined. The taxonomic history of this species was compiled using the taxonomic literature and online databases POWO [3] and IPNI [41]. The morphological characteristics, cultivation areas, habitats, pollinators, growing periods, phenology, vernacular names, and uses were described from our observations during field work and from label information on the specimens examined.
The preparation of plant samples was for anatomical observation. Transverse sections of the stems and the leaf lamina were through the midribs. The stem and leaf samples were dehydrated in an increasing ethanol concentration series of 30%, 50%, 70%, 95%, and absolute ethanol, embedded in paraffin, sectioned with a rotary microtome at 16-20 µm thickness with Haupt ′ s adhesive affixing paraffin sections to slides, stained with safranin and fast green, cleared with xylene, and mounted in DePeX mounting media. Leaf epidermal preparations were made by peeling and mounting on slides. The anatomical characteristics were investigated and recorded photographically with an Olympus BX53 microscope and an Olympus DP74 microscope digital camera at the Department of Botany, Faculty of Science, Kasetsart University (KU). The anatomical terminologies follow those in the study by Metcalfe and Chalk [37].
The samples of pollen grains were taken from the herbarium specimen collected from Mae Hong Son and Chiang Mai provinces (C. Ngernsaengsaruay et al. Ps01-06012023, C. Ngernsaengsaruay et al. Ps02-06012023, C. Ngernsaengsaruay et al. Ps03-06012023, C. Ngernsaengsaruay et al. Ps04-06012023, C. Ngernsaengsaruay et al. Ps05-04032023, and C. Ngernsaengsaruay et al. Ps06-04032023). They were examined and recorded photographically with an Olympus BX53 microscope and an Olympus DP74 microscope digital camera. Materials were prepared for scanning electron microscopy (SEM) at the Scientific Equipment Centre, Faculty of Science, KU by mounting pollen grains on stubs using doublesided sellotape, sputter-coating them with gold and examining them using an FEI Quanta 450 SEM (Hillsboro, OR, USA) at 15.00 KV. The characteristics of fifty pollen grains (polarity, symmetry, shape, size, aperture, exine thickness, and sculpturing) were examined and measured, following Erdtman [42,43] and Simpson [44]. The pollen morphology terminologies follow those of Punt et al. [45].

Conclusions
Opium poppy is illegal in most parts of the world, including Thailand; as such, information about opium poppy is mostly lacking and its characteristics poorly known. We have obtained permission to study and support from the Narcotics Crop Survey and Control Institute, ONCB, Ministry of Justice. In this research project, we provide the morphology, taxonomy, anatomy, and palynology of Papaver somniferum. As a result, we update detailed morphological descriptions for this species, which is more than the information in previous studies in Thailand and neighboring countries. With proven benefits of opium poppy already reported, the Office of the Narcotics Control Board, Thailand, plans to study opium poppy for further usage in the pharmaceutical industry.
The species can be characterized as a glabrous and glaucous herb, with unlobed or pinnately lobed leaves and an amplexicaul base; with variations in color and morphological characteristics of petals; and white filaments, occasionally purple with a white basal part, broadened at the apical part.
From our study, we found that the number of seeds per capsule of Papaver somniferum has a range of 593-4685. We realized that the number of seeds per capsule depends on the size of fruits and number of carpels.
Even when Papaver somniferum is notoriously known as a highly addictive non-synthetic narcotic, its usage in deriving pain killers (morphine) should not be ignored. The seeds are used in food, oils, and pharmaceuticals to treat inflammation, are cardiotonic, mildly astringent, analgesic, and sedatives. As per international law, the usage of P. somniferum should be strictly controlled and only used under supervision. However, a complete ban on the cultivation of P. somniferum would be counterproductive, given its high potential usage. Regarding the variation in the morphology of P. somniferum even in the same location, this might suggest that it can have various pharmaceutical properties.
Funding: This research was funded by the Office of the Narcotics Control Board, Ministry of Justice.
Data Availability Statement: All relevant data can be found within the manuscript.
Bhuchaisri for the line drawings, and Pattarin Nunthamontree and Atcharaphan Saengbuapuean for photos of the herbarium specimens of Papaver somniferum deposited in QBG and BKF, respectively.